other: Test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Justification for type of information:

Please refer to Category Document

Principles of method if other than guideline:

The objective of this study is to evaluate the ability of MIBK, under exposure conditions of the 2-year chronic bioassay, to induce specific measures of alpha2µ-nephropathy (alpha2µ-N) such as histological lesions associated with the accumulation of alpha2µ globulin, increased renal concentration of alpha2µ globulin, and sustained renal cell proliferation in the kidneys of male but not female rats. In addition, the ability of MIBK to bind reversibly to alpha2µ globulin was investigated, fulfilling another criterion that supports the alpha2µ-N MoA. Identification of the MoA for MIBK-induced renal toxicity and carcinogenesis is important for assessing the human health risks from exposure to MIBK.

GLP compliance:

yes

Remarks:

Both the inhalation MIBK study and the o-limonene oral dose study were conducted in accordance with U.S. EPA's GLP from animal receipt through euthanasia but not including dose formulation for o-limonene.

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION- Exposure apparatus: 1 m3 stainless steel and glass inhalation cages (Hazelton H1000 Lab Products, Seaford, DE)- Method of holding animals in test chamber: whole-body exposure- Source and rate of air: no data- Method of conditioning air: no data- System of generating particulates/aerosols: a liquid metering pump (Fluid Metering Inc., FMI) was used to direct liquid MIBK into a J-tube generator.The FMI pump was calibrated and set to the nominal flow rate expected for each exposure concentration. Nitrogen flowed upward through the J-tube in all concentration groups at a flow rate of approximately 25 L/min. The MIBK vapor flow was introduced counter current to the HEPA-filtered chamber airtlow to facilitate mixing of the MIBK vapor with dilution air.- Temperature, humidity: 20-25°C, 30-70%- Air flow rate: approximately 250 L/min- Air change rate: 15 air changes each hour- Method of particle size determination: not relevant for vapor- Treatment of exhaust air: no data

The concentration of the test atmosphere in each chamber was analyzed using a Hewlett Packard Series lI 5890 gas chromatograph (Agitent Technologies, Inc., Palo Atlo, CA) equipped with a flame ionization detector (FID) and 10-port sampling valve (VIVI Valco Instruments, Houston, TX). The 10-port sampling valve rotated through a series of positions, which sampled from the exposure chambers and the control chamber. The gas chromatography analysis was timed so that the valve rotated through all 10 positions in 30 min during the exposure period.

Three and a half days prior to euthanasia, at both 1- and 4-weeks, rats were subcutaneously implanted with an osmotic pump containing 5-bromo-2-deoxyuridine (BrdU) to measure cell proliferation. At study termination, body weights were recorded and animals euthanized to harvest kidneys.

Examinations:

Rats in both the MIBK inhalation study and the o-limonene positive control study were euthanized with an intraperitoneal injection of sodium pentobarbital ( 100 mg/kg) and then exsanguinated from the abdominal aorta. The kidneys were removed and prepared for histopathology and biochemical endpoints.

Positive control:

o-Limonene is a chemical used as a positive control for measures of α2µ-N since it fulfills all of the criteria outlined by IARC (1999) to characterize this response. ln this positive control study, twelve male rats were randomly allocated to one of two dose groups. Rats were administered either corn oil (vehicle control) or o-limonene at a dose of 300 mg/kg/day (5 mL/kg) byoral gavage for 4 consecutive days and euthanized ~24 h following the last dose.

Details on results:

There was an exposure-related increase in all measures of α2µ nephropathy in male, but not female rat kidneys. The hyaline droplets present in male rat kidney stained positively for α2µ. The changes in HDA and α2µ concentration were comparable to d-limonene, an acknowledged inducer of α2µ nephropathy. In a separate in vitro study using a two-compartment vial equilibration model to assess the interaction between MIBK and α2µ, the dissociation constant (Kd) was estimated to be 1.27×10(-5)M. This Kd is within the range of other chemicals known to bind to α2µ and cause nephropathy.

Terminal body and kidney weights

There were no statistical differences in the final body weights of animals exposed to MIBK compared to concurrent controls. There was a significant increase in absolute kidney weights in male and female rats exposed to 900 or 1800 ppm MIBK for 1-and 4-weeks compared to the concurrent control along with an exposure-related increase at the 4-week exposure period.

Histopathology

A trace of intertubular mineralization was observed in the cortex of H&E stained kidney section from both male and female rats. Chronic progressive nephropathy (CPN) is a background lesion in control rats that is identifiable at 2-4 months of age in male rats of susceptible strains such as the F344 (Hard et al., 2013 ). In this study the mean grade of CPN in rats not exposed to MIBK was 0.38-0.50. Exacerbation of CPN was not observed after 1 week of MIBK exposure or administration of the positive control o-limonene. However, exacerbation of CPN was slightly increased in male rats exposed to 900 and 800 ppm MIBK for 4 weeks. CPN was not exacerbated in female rats following exposure to MIBK for either 1- or 4-weeks.

Accumulation of protein (hyaline) droplets associated with MIBK exposure was observed in proximal convoluted tubules of all MIBK-exposed male, but not female rats evaluated in both H&E (viewed under fluorescence) and MH-stained kidney sections. With increasing MIBK exposure concentration, in both1- and 4-weeks, there was increasing prominence of hyaline droplets in terms of size of droplet tracts, droplet pattern disruption observed with fluorescence microscopy as well as withincreasing intensity of autofluorescence. On a 0-16 scale, an exposure-related increase in HDA was observed in 1 week, ranging from grade 1 in the control male rats up to grade 10.75 in the high exposure male rat group (1800 ppm) (Fig. 3). The male rats exposed to MIBK for 4 weeks showed the same trend as the animals exposed for 1 week. HDA was also prominent in the o-limonene positive control group with a mean grade of 12 after 1 week of exposure. The sections of kidney stained with anti-a2µ antibody showed the protein droplets accumulating in male rats exposed to MIBK oradministered o-limonene were positive for a2µ, whereas there was less staining in control male rats kidney and no staining in female rats. Although there was an absence of well-formed granular casts in any exposure group, 6/8 males exposed to 1800 ppm MIBK for 4 weeks had solitary tubules at the junction of the outer and inner stripes of the outer medulla containing eosinophilic granular debris. These were consistent with precursors of granular casts described by Hard (2008).

Renal concentration of a2µ and total protein

The concentration of a2µ was measured in the kidney homogenate of male and female rats exposed to MIBK or male rats administered corn oil or the positive control o-Iimonene. MIBK caused an exposure related increase in concentration of a2µ in themale rats kidneys at both 1- and 4-weeks of exposure (Fig. 5A and C). Although, the magnitude of the increase was not as significant in the positive control o-limonene administered male rats (Fig. SA), it was statistically significant compared to the concurrent control at ail MIBK exposure concentrations. Total protein was not changed in male rats exposed to MIBK, but there was a significant increasefollowing o-limonene administration (Fig. 5B and D). The concentration of a2µ in female rat kidneys was 250-fold lower compared to male rats. Although there was a significant decrease in the concentration of a2µ in female rats exposed to 1800ppm MIBK compared to the concurrent control, considering the low level of a2µ in the female kidneys it is most likely that this change is not biologically significant and further demonstrates that MIBK does not increase a2µ in the kidneys of female rats (Fig. 6A).

Renal cell proliferation

Following 1 week of MIBK exposure, there was a statistically significant increase in the percent of cells labeled with BrdU (LI) in male rats exposed to 450 ppm MIBK compared to the concurrent control, but not following 900 or 1800 ppm (Fig. 7). The increase in the LI was more apparent in male rats following 4 weeks of MIBK exposure with a significant increase at ail exposure concentrations compared to the concurrent controls. A significant exposure related increase in LI was observed at bath 1 and 4 weeks in male rats. Although there were no changes in the LI in the female rats between each exposure group and concurrent control at either 1- or 4-weeks of MIBK exposure, there was a significant exposure­related increase following the 1-week exposure period. Considering the data at both exposure periods, this change does not appear to have any biological significance.

The counts of mitotic figures in cortical proximal tubule cells were approximately 10 times higher in males rats exposed to 1800ppm MIBK for 1- or 4-weeks compared to their concurrent controls (Table 4). All of the mitotic figures counted were in proximal convoluted tubules and many were in tubules containinghyaline protein droplets. There was no increase in mitotic counts in high-dose females at 1- or 4-weeks of exposure.

In vitro binding of MIBK to a2µ

Total protein concentration was determined to be 145.4 ±14.6 mg/g and 147.6 ± 7.8 mg/g total kidney in male and female rats, respectively. There was a concentration dependentdecrease in PC in male, but not female kidney tissue suggesting that the uptake of MIBK into male rat tissue is only dependent on solubility alone (Fig. 9). The PC of MIBK in male rat kidney tissue was decreased after the addition of oLO, a known ligand for a2µ, providing evidence that MIBK is displaced from binding to a2µ (Fig. 9). Table 5 provides the 2-compartment model parameter values achieved mathematically to describe the uptake of MIBK into the kidney tissue based on solubility and binding to a2µ. Using this model and the data presented in Fig. 9, aKdwas estimated in the range of 1.27 x 10-5 M to describe binding of MIBK to a2µ. This 2-compartment modeling approach provides an indirect method to assess chemical binding to a2µ with volatile chemicals and was used previously for methyl tertiary butyl ether (MTBE) (Poet and Borghoff, 1997).

Conclusions:

These studies provide the data necessary to demonstrate the ability of MIBK to induce exposure-related male rat specific changes in measures of a2µ-N (protein droplet accumulation, staining of the protein droplets for a.2µ, sustained renal cell proliferation, and an increased concentration of a2µ along with providing evidence that MIBK binds reversibly to a2µ, the initiating event in a2µ-N. The strength of these data is supported by previous studies that have contributed to the characterization of MIBK as an inducer of a2µ-N (Dodd et al., 1982; Phillips et al., 1987; Nemec et al., 2004; Stout et al., 2008; Borghoff et al., 2009) meeting all of the IARC criteria (IARC, 1999) for characterization of this MoA. Together these studies provide the weight of evidence that the MIBK induced male rat renal tumors are not relevant to humans.

Executive summary:

Chronic exposure to methyl isobutyl ketone (MIBK) resulted in an increase in the incidence of renal tubule adenomas and occurrence of renal tubule carcinomas in male, but not female Fischer 344 rats. Since a number of chemicals have been shown to cause male rat renal tumors through the α2µ nephropathy-mediated mode of action, the objective of this study is to evaluate the ability of MIBK to induce measures of α2µ nephropathy including renal cell proliferation in male and female F344 rats following exposure to the same inhalation concentrations used in the National Toxicology Program (NTP) cancer bioassay (0, 450, 900, or 1800 ppm). Rats were exposed 6h/day for 1 or 4 weeks and kidneys excised approximately 18h post exposure to evaluate hyaline droplet accumulation (HDA), α2µ staining of hyaline droplets, renal cell proliferation, and to quantitate renal α2µ concentration. There was an exposure-related increase in all measures of α2µ nephropathy in male, but not female rat kidneys. The hyaline droplets present in male rat kidney stained positively for α2µ. The changes in HDA and α2µ concentration were comparable to d-limonene, an acknowledged inducer of α2µ nephropathy. In a separate in vitro study using a two-compartment vial equilibration model to assess the interaction between MIBK and α2µ, the dissociation constant (Kd) was estimated to be 1.27×10(-5)M. This Kd is within the range of other chemicals known to bind to α2µ and cause nephropathy. Together, the exposure-related increase in measures of α2µ nephropathy, sustained increase in renal cell proliferation along with an indication of reversible binding of MIBK to α2µ, support the inclusion of MIBK in the category of chemicals exerting renal effects through a protein droplet α2µ nephropathy-mediated mode of action (MoA).

Male and female rats (4/sex/treatment group) were administered corn oil (vehicle control) or MIBK (1000 mg/kg bw/day) and another group of male rats (n=4) were administered d-limonene (positive control; 300 mg/kg bw/day) for 10 consecutive days by oral gavage. Approximately 24 h after the final dose the kidneys were excised and the left kidney prepared and evaluated for histological changes including protein (hyaline) droplet accumulation, immunohisto-chemical staining for α2μ-globulin (α2μ) nephropathy (α2μ-N), and proliferating cell nuclear antigen (PCNA) to quantitate renal cell proliferation. The right kidney was prepared for quantitation of total protein and α2μ using an enzyme-linked immunosorbent assay (ELISA).

MIBK elicited an increase in protein droplets, accumulation of alpha2µ, and renal cell proliferation in male, but not female rats, responses characteristic of alpha2µ-mediated nephropathy. MIBKproduced identical histopathological changes in the male rat kidney when compared to d-limonene, an acknowledged inducer of alpha2µ-nephropathy except that the grade of severity tended to be slightly lower with MIBK. MIBK did not induce any effects in female rats.

Exposure to MIBK or d-limonene did not affect body weight gain, terminal body weights, and total kidney weight over the length of this study; however, there was a slight increase in absolute kidney weights in female rats administered MIBK. The kidney:body weight ratio was increased slightly in male and female rats as compared to their respective controls. MIBK-treated male rats showed an identical range of changes in the kidney as compared to the changes noted in the kidneys from d-limonene treated male rats. The severity of droplet accumulation was graded as mild in 3/4 male rats, and moderate in 1/4 rats. Thus, the various effects appeared at a slightly lower grade than with d-limonene. All of the female rat kidneys were judged to be within normal limits with no renal alterations produced by MIBK administration. No histopathological evidence explaining the slightly increase in absolute and relative kidney weights was evident for female rats. All male rats treated with 1000 mg/kg bw/day MIBK had positive Mallory’s Heidenhain (MH) staining similar to that seen in kidney sections of d-limonene treated male rats with respect to the disruption of the normal pattern of staining and the presence of crystal-like aggregates. The kidneys sections from MIBK-treated female rats were negative for MH-stained droplets in proximal tubule cytoplasm. Immunohistochemical staining of kidney sections for α2μ localized the protein to the renal cortex of control male rats. The staining in kidney sections from both MIBK and d-limonene treated male rats was more intense and occupied a greater area of the renal cortex than that of control rats. Due to uneven staining on the kidney sections, semi-quantitative grading of these sections was difficult. No positive staining for α2μ was observed in the kidneys from control or MIBK administered female rats. A statistically significant threefold increase in renal cell proliferation was reported in male rats administered MIBK, but not in female rats compared to their respective controls.

Executive summary:

Male F-344 rats were administered corn oil (vehicle control), d-limonene (positive control, 300mg/kg), or MIBK (1000mg/kg) and female F-344 rats corn oil (vehicle control) or MIBK for 10 consecutive days by oral gavage. Approximately 24h after the final dose the kidneys were excised and the left kidney prepared and evaluated for histological changes including protein (hyaline) droplet accumulation, immunohistochemical staining for alpha2µ-globulin (alpha2µ), and proliferating cell nuclear antigen (PCNA) to quantitate renal cell proliferation. The right kidney was prepared for quantitation of total protein and alpha2u using an ELISA. MIBKelicited an increase in protein droplets, accumulation of alpha2µ, and renal cell proliferation in male, but not female rats, responses characteristic of alpha2µ-mediated nephropathy. MIBKproduced identical histopathological changes in the male rat kidney when compared to d-limonene, an acknowledged inducer of alpha2µ-nephropathy except that the grade of severity tended to be slightly lower with MIBK. MIBK did not induce any effects in female rats. Therefore, renal histopathology, along with the other measures of alpha2µ accumulation, provides additional weight of evidence to support the inclusion of MIBK in the category of chemicals exerting renal effects through a alpha 2µ-nephropathy-mediated mode-of-action.

Male B6C3F1 mice, six control and six treated, were exposed to target concentrations of 0 or 1800 ppm MIBK vapors via inhalation (whole-body) for six hours/day for 7 consecutive days. Liver-specific clinical chemistry, histopathology, targeted gene expression, cytochrome P450 enzymatic activity, and hepatocellular proliferation were measured. Animals were sacrificed on the morning following their final exposure.

TEST ATMOSPHERE- Brief description of analytical method used: The various vapor concentrations of MIBK were generated using a glass J-tube method (Miller et al., 1980). Liquid test material was pumped into the glass J-tube assembly and vaporized by nitrogen gas passing through the bead bed of the glass J-tube (20 liters per minute). The nitrogen was heated with a flameless heat torch (FHT-4, Master Appliance Corporation, Racine, Wisconsin) to the minimum extent necessary to vaporize the test material. The generation system was electrically grounded and the J-tubes were changed as needed. The nitrogen carrier gas with vaporized test material was mixed and diluted with filtered supply air to achieve the desired test chamber concentration. Animals in the control group were exposed to the same volume percent of nitrogen as the MIBK exposed animals.

- Samples taken from breathing zone: yes

VEHICLE (if applicable)-Not applicable

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The chamber concentrations of MIBK, measured approximately in the center of the breathing zone of the animals, was determined at least once per hour with a Miran 1A infrared (IR) spectrophotometer (Foxboro/Wilks, South Norwalk, Connecticut). The IR spectrophotometer was calibrated and a standard curve compiled prior to the start of the study, using air standards prepared by vaporizing measured volumes of MIBK into Tedlar® sample bags (Series 233, SKC, Eighty Four, Pennsylvania) along with metered volumes of filtered air. The analytical concentration during the exposure was interpolated by the CAMILE TG Data Acquisition and Control System using the standard curve. The analytical system was checked prior to and after each exposure with a MIBK standard gas-bag of known concentration. Output from the infrared spectrophotometer was monitored by the CAMILE Data Acquisition and Control System, displayed, logged to memory, and printed for inclusion in the study file. Prior to exposure of animals, the distribution of vaporized test material was determined from five sample points in the breathing zone and the normal sampling point (reference point) in the chamber in order to ensure that a uniform distribution of vapor is present throughout the breathing zone of the animals. The daily nominal concentration of the test material was calculated based on the amount of test material used divided by the total airflow through the chamber each day.

Duration of treatment / exposure:

7 days

Frequency of treatment:

6 hours/day

Post exposure period:

None

Remarks:

Doses / Concentrations:1800 ppmBasis:analytical conc.

No. of animals per sex per dose:

6

Control animals:

yes, sham-exposed

Examinations:

Body weight/body weight gains; Cage-side clinical observations; Clinical chemistry (at necropsy); Liver weights; Targeted gene expression analysis of the upper third of the left liver lobe; Histopathological examination and BrdU proliferation analysis of middle third of the left liver lobe; Sample of duodenum as a positive control for proliferation analysis; Enzyme activity analysis (lower portion of the left lobe and the medial lobe of the liver).

Positive control:

None

Details on results:

Treatment-related findings included very slight hepatocytes hypertrophy with increased cytoplasic eosinophilia in the centrilobular/midzonal regions of the hepatic lobule which were consistent with increased smooth endoplasmic reticulum and induction of cytochrome P450 enzymes. CYP2B10transcript levels increased 4-fold andCYP4A10decreased 5.56-fold. This was verified by increased CYP2B10 enzyme activity (PROD) and hepatocyte proliferation. These responses are commonly observed following activation of constitutive androstane receptor (CAR) and indicate that MIBK may be an agonist ligand for CAR in mice and share a similar mode of action to that of Phenobarbital in mice. The study authors noted that this mechanism of action is not relevant to humans.

No treatment related effects were observed for clinical signs, body weights, liver weights, or clinical chemistry measurements in MIBK exposed animals. Treatment-related histopathologic changes in the liver consisted of very slight hepatocyte hypertrophy (enlargement) with altered tinctorial properties (increased cytoplasmic eosinophilia) in the centrilobular/midzonal regions of the hepatic lobule. These changes were consistent with possible increased smooth endoplasmic reticulum and induction of cytochrome P450 enzymes. Significant transcript level alterations were observed for CYP2B10 (increased 4-fold) and CYP4A10 gene expression (decreased 5.56-fold). No changes were observed in CYP1A1 or CYP3A11 gene expression levels. Significant inductions of CYP2B10 enzyme activity (PROD) and hepatocellular proliferation were also observed. These responses are commonly observed following activation of constitutive androstane receptor (CAR) and are typical for phenobarbital (PB)-like compounds. Together with literature observations, these findings indicate that MIBK may be an agonist ligand for CAR and share a similar mode of action to that of phenobarbital.

Executive summary:

In a non-guideline and non-GLP study, the effects of methyl isobutyl ketone (MIBK) on the mouse liver were evaluated (Geter, 2009).Male B6C3F1 mice were implanted with 5-bromo-2’deoxyuridine (BrdU) pumps and then exposed to 0 or 1800 ppm (n=6/group) of MIBK via whole-body inhalation for 6 hours/day for 7 days.In-life assessments included clinical signs and body weights.Mice were euthanized and assessed for clinical chemistry, gene expression analysis of the upper third of the left liver lobe, liver histopathological examination and BrdU proliferation analysis, and liver enzyme activity.There were no treatment-related effects noted for clinical signs, body weights, liver weights, or clinical chemistry assessments.Treatment-related findings included very slight hepatocytes hypertrophy with increased cytoplasic eosinophilia in the centrilobular/midzonal regions of the hepatic lobule which were consistent with increased smooth endoplasmic reticulum and induction of cytochrome P450 enzymes.CYP2B10transcript levels increased 4-fold andCYP4A10decreased 5.56-fold.This was verified by increased CYP2B10 enzyme activity (PROD) and hepatocyte proliferation.These responses are commonly observed following activation of constitutive androstane receptor (CAR) and indicate that MIBK may be an agonist ligand for CAR in mice and share a similar mode of action to that of Phenobarbital in mice.The study authors noted that this mechanism of action is not relevant to humans.

MIBK induced hepatic effects are consistent with a phenobarbital-like MOA where the initiating events are activation of the Car and Pxr nuclear receptors leading to rodent liver tumors.

Executive summary:

Methyl isobutyl ketone (MIBK) is widely used in the coatings industry. A 2007 NTP study identified that MIBK induces liver tumors in B6C3F1 male (M) and female (F) mice; however, MIBK lacks genotoxicity. Previous studies suggested that the mode of action (MOA) by which MIBK induces hepatocellular tumors in mice is through phenobarbital-like nuclear receptor associated activation. To further investigate the MOA for MIBK-induced murine liver tumors, M and F B6C3F1, C57BL/6, and Car/Pxr Knockout (KO) mice were exposed to either 0 or 1800 ppm MIBK for 6 h/d, 5 d/w for a total of 10 days. On day 1, mice were implanted with osmotic mini-pumps containing 5-Bromo-2-deoxyuridine (BrdU) 1 h following exposure and humanely euthanized 1 to 3 h following the final exposure. B6C3F1 and FC57BL/6 mice had statistically significant increases in liver weights compared to controls that corresponded with hepatocellular hypertrophy and increased mitotic figures. Hepatocellular proliferation data indicated induction of S-phase DNA synthesis in B6C3F1 and C57BL/6 mice exposed to 1800 ppm MIBK compared to control, no increase was observed in MIBK exposed Car/Pxr KO mice. Liver gene expression changes indicated a maximally-induced Cyp2b10 (Car-associated) transcript and a slight increase in Cyp3a11(Pxr-associated) transcript in B6C3F1 and C57BL/6 mice exposed to 1800 ppm MIBK compared to controls, but not in Cyp1a1 (AhR associated) or Cyp4a10 (PPAR-α-associated) transcripts. Car/Pxr KO mice showed no evidence of activation of AhR, Car, Pxr or PPAR-α nuclear receptors via their associated transcripts. MIBK induced hepatic effects are consistent with a phenobarbital-like MOA where the initiating events are activation of the Car and Pxr nuclear receptors leading to rodent liver tumors.

Description of key information

A study in mice indicated that methyl isobutyl ketone (MIBK) may be an agonist for the constitutive androstane receptor (CAR) in liver mice. This was evaluated following inhalation exposure to 1800 ppm MIBK for 7 days. The study authors noted that this mechanism of action for liver carcinogenicity is not relevant to humans. A study in rats, demonstrated that MIBK may exert effects on the male rat kidney via an alpha-2u-globulin mechanism. As well, this mechanism of action for kidney carcinogenicity, which is species and male specific, is not relevant to humans.

Additional information

The key studies for this group of substances (DIBC, DIBK, MIBK and MIBC) are two 2 -year carcinogenicity studies in mice and rats using MIBK. These studies showed the potential for MIBK to produce kidney tumours in rats and liver tumours in mice. Given the absence of any genotoxicity for this group of substances it was considered that the mode of action involved a non-genotoxic, threshold mechanism. In addition, the relevance of these tumour types to humans has been questioned due to the data available on the potential mode of action. The below studies were conducted to understand whether the mode of action for MIBK carcinogenicity in liver and kidney was in fact relevant for humans. In addition to these studies, additional data are also being generated to provide further support for the mechanism of tumour formation and its lack of relevance to humans.

Given the structural similarities and consistent toxicological profiles within this group of substances it is considered plausible that all members could produce toxicity in the livers and kidneys that may ultimately result in tumor formation in chronic studies. However, it therefore follows that their structural similarities and common metabolic pathways would also support a common mode of action, and thus the data that support a lack of human relevance for MIBK carcinogenicity also support the lack of relevance for the other members of the group.

The development of kidney tumors in male rats in association with chemically inducedα2µ-globulin nephropathy is mechanism that is not considered to be a predictor of carcinogenic risk to humans by the IARC or the US EPA (US EPA, 1991; Hard et al., 1993; Swenberg & Lehman-McKeeman, 1999). The lack of relevance of the α2µ-globulin mechanism for the evaluation of carcinogenic risk is based on the absence of the production of an analogous protein in humans. Strict scientific criteria have been outlined to establish the role ofα2µ-globulin-associated nephropathy in renal carcinogenesis in male rats (Swenberg & Lehman-McKeeman, 1999), and were used to determine the plausibility of anα2µ-globulin associated nephropathy based on a studies that have been carried out with subchronic and chronic exposures to methyl isobutyl ketone.

1. Lack of genotoxic activity (agent and/or metabolite) based on an overall evaluation of in-vitro and in-vivo data

A battery of genotoxicity assays with MIBK published in O’Donoghue et al. (1988) yielded mostly negative responses. MIBK did not induce revertant point mutations in five Salmonella tester strains (TA98, TA100, TA1535, TA1537, and TA1538), either in the presence or absence of Aroclor 1254-induced rat liver microsomal enzymatic activation. Mutant frequencies were also not affected in the L5178Y TK+/- mouse lymphoma mutagenesis assay in the presence of Aroclor-induced rat liver S-9. No dose-response relationship was observed in cultures exposed to MIBK in the absence of exogenous metabolic activation, although mutation frequency was significantly elevated in three out of six of the MIBK-treated cultures in the absence of S-9 metabolic activation. MIBK was also negative in the unscheduled DNA synthesis assay in rat primary hepatocytes cultures and in the in vivo micronucleus cytogenetic assay in mice administered MIBK intraperitoneally at 0.73 mL/kg (the dose level selected as the LD20 on the basis of a preliminary toxicity study). The following additional set of genotoxicity assays also yielded negative results: reverse mutation assays in five strains ofSalmonella typhimuriumand three strains ofEscherichia coliand gene mutations inSaccharomyces cerevisiae, all both in the presence and absence of exogenous metabolic activation and a structural chromosome damage assay in cultured rat liver cells (Brooks et al., 1988).

MIBC was not mutagenic in bacterial reverse mutation assays (Shimizu et al., 1985; Clare, 1984) and mammalian cells (L5178Y) in vitro with or without metabolic activation (Sire 2010b). In a mammalian cell cytogenetic assay in rat liver cells, MIBC was negative with and without metabolic activation (Clare, 1984).

Thus, this criterion is met.

2. Male rat specificity for nephropathy and renal tumorigenicity induction of the characteristic sequence of histopathological changes in shorter-term studies, of which protein droplet accumulation is obligatory

Groups of six male and six female rats were exposed for 6 hrs/day, 5 days/week, for 9 days to measured concentrations of 0, 101, 501, or 1996 ppm (0, 410, 2053, or 8178 mg/m3) MIBK (Dodd et al., 1982). Groups were evaluated for changes in clinical signs, body weight, organ weights (liver, lungs, kidneys, and testes), ophthalmology, gross pathology, and histopathology. The only exposure-related effects observed were periocular wetness in rats exposed to 8178 mg/m3, increased relative liver weights in male rats at 2053 and 8178 mg/m3 and in female rats, increased kidney weights in male rats, and hyaline droplet degeneration in kidneys of male ratsexposed to 2053 or 8178 mg/m3, with epithelial regeneration of proximal convoluted tubules in the high-exposure group. No effects of any kind were observed in the 410 mg/m3 exposure group.

In a 90-day inhalation study in rats, groups of 14 male and 14 female Fischer 344 rats were exposed to measured mean concentrations of 0, 50, 252, and 1002 ppm (0, 205, 1033, and 4106 mg/m3) MIBK for 6 hrs/day, 5 days/week, for 14 weeks and sacrificed the animals following their final exposure day (Dodd and Eisler, 1983; Phillips et al., 1987). The following endpoints were evaluated: clinical signs, body weights, organ weights (kidneys, heart, liver, lungs, and testes), urinalysis, haematology, serum chemistry (including glucose and hepatic enzyme levels), complete gross pathology, targeted histopathology (nasal cavity, trachea, liver, kidneys, and lungs) in all animals and complete histopathology in control and high-exposure groups…//… Urine glucose was significantly increased in male rats at 1033 mg/m3 (+37%) and 4106 mg/m3 (+55%) and in female rats at 4106 mg/m3 (+26%). Significantly increased urine protein (+28%) was also observed in male rats at 4106 mg/m3.The only renal histological lesion observed was hyaline droplet formation in all male rats; the severity of the lesion generally increased with exposure level. In conclusion, other than the male rat kidney effect, exposure of male and female rats to MIBK at levels up to 1000 ppm for 14 weeks was without significant toxicological effect.

These studies indicate the induction of hyaline protein droplets in shorter-term studies.α2µ-globulin protein droplets were also specifically identified and characterized further in the studies of Borghoff et al. (2009 and 2015).

Thus, this criterion is met.

3. Identification of the protein accumulating in tubule cells asα2µ-globulin

The effects of MIBK exposure on Fischer 344 rat kidneys were assessedunder exposure conditions used in the 2-year MIBK chronic bioassay (Borghoff et al., 2015). Rats were exposed 6h/day for 1 or 4 weeks and kidneys excised approximately 18h post exposure to evaluate hyaline droplet accumulation (HDA), α2µ staining of hyaline droplets and to quantitate renal α2µ concentration. There was an exposure-related increase in all measures of α2µ nephropathy in male, but not female rat kidneys. The hyaline droplets present in male rat kidney stained positively for α2µ. The changes in HDA and α2µ concentration were comparable to d-limonene, an acknowledged inducer of α2µ nephropathy Although this study was of short duration, it was designed to provide additional support to characterize MIBK as an inducer ofα2µ-nephropathy, a MoA associated with a low incidence of kidney tumors in male, but not female rats.

The effects of MIBK oral exposure on Fischer 344 rat kidneys were assessed (Borhoff et al., 2009). Male and female rats were administered corn oil, 1000 mg/kg bw/day MIBK, or d-limonene (positive control) for 10 consecutive days by oral gavage (4/sex/group).Rats were euthanized 24 hours after the last dose, and the left kidney evaluated for histological changes in hyaline droplet accumulation, α-2µ-nephropathy, and proliferating cell nuclear antigen.The right kidney was assessed for total protein and α2µ-globulin using an enzyme-linked immunosorbent assay (ELISA).There were no changes in body weight gain, terminal body weights, or total kidney weight.The kidney/body weight ratio was increased slightly in male and female rats compared to controls.Changes observed in MIBK-treated male rats were similar or slightly milder than those observed in d-limonene-treated rats.Findings included mild to moderate hyaline droplet accumulation, positive Mallory’s Heidenhain staining, more intense alpha-2u-globulin staining compared to untreated controls, and a statistically significant increase in renal cell proliferation.There were no changes noted in any of the females administered MIBK.These results provide support that MIBK exerts renal effects in male rats through an alpha-2u-mediated mechanism.

Thus, this criterion is met.

4. Reversible binding of the chemical or metabolite toα2µ-globulin

In anin vitrostudy using a two-compartment vial equilibration model to assess the interaction between MIBK andα2µ, the dissociation constant (Kd) was estimated to be 1.27×10(-5)M. This Kd is within the range of other chemicals known to bind toα2µ and cause nephropathy(Borghoff et al., 2015).

The last criterion is the reversible binding of the chemical or metabolite toα2µ-globulin, which was not shown direct in any of the studies. One study showed reversibility of the adverse effects in the kidney after withdrawal of MIBK. The Wright-Patterson Air Force Base Aerospace Medical Research Laboratory (MacEwenet al., 1971) conducted a subchronic inhalation toxicity study in male Wistar albino rats that were exposed to 410 mg/m3MIBK vapour [100 ppm] for 90 days in an altitude chamber. The untreated control group was maintained in a separate altitude chamber. Statistically significant increases in liver and kidney weights and organ-to-body weight ratios for these tissues were noted in exposed rats. Microscopic examination of the kidneys revealed hyaline droplet degeneration of the proximal tubules (with occasional foci of tubular necrosis) in all of the exposed rats, including those that were removed from the inhalation chamber after 15, 22, 28, 71 and 85 days. The authors noted a trend towards a linear progression of hyaline droplet degeneration during exposure, but this pattern was not seen in all treatment groups. Moreover, the hyaline droplets appeared to increase in size with time. This observation was thought to have resulted from the coalescence of smaller droplets. Microscopic examination of rat kidneys removed after 15 days of exposure indicated a gradual reversion of tubular damage with time. Kidney damage was completely reversed in rats observed up to 60 days after exposure. Recovery MIBK-induced kidney lesions was also noted in rats that were serially killed to study reversibility after 90 days of exposure. However, recovery was not as rapid as that noted in animals exposed for shorter periods. A weakness of the study was the exclusion of female rats. The study showed the reversibility of effects that could be attributed toα2µ-globulin nephropathy.

The effects of MIBK exposure on Fischer 344 rat kidneys were assessedunder exposure conditions used in the 2-year MIBK chronic bioassay (Borghoff et al., 2015). Male and female rats were exposed 6h/day for 1 or 4 weeks and were subcutaneously implanted with an osmotic pump containing 5-bromo-2-deoxyuridine (BrdU). Kidneys were excised approximately 18h post exposure to measure cell proliferation by immunohistochemical staining for BrdU.A significant exposure related increase in cells labeled with BrdU (LI) was observed at 4 weeks in male rats. The counts of mitotic figures in cortical proximal tubule cells were approximately 10 times higher in males rats exposed to 1800ppm MIBK for 1- or 4-weeks compared to their concurrent controls.

The histological endpoints identified (i.e., protein droplets,α2µaccumulation and even cell proliferation) are responses that occur early in the development ofα2µ-nephropathy. A dose-response between these early lesions and renal tumors is difficult to demonstrate especially when the incidence of tumors is low and in the case of MIBK the two mid doses show a similar tumor response. As presented in theIARC MIBK evaluation (IARC, 2012), there was a correlation with linear mineralization and incidence of tumors that provided support for the association betweenα2µ-nephropathy and male rat renal tumors. ln the study of Borghoff et al. (2015), early measures ofα2µ-nephropathy shown that there is an exposure-related correlation between renalα2µconcentration and HDA (Fig. 10A) as well as HDA and the labelling index (LI) in the renal cortex of male rats (Fig. 10B), supporting an association between these events.

Thus, this criterion is met.

Conclusion

All of the IARC criteria for characterization of the MoA are met. Together these studies provide the weight of evidence that the MIBK induced male rat renal tumors are not relevant to humans.

In the NTP bioassay (2007), significant positive trends were reported for hepatocellular adenomas in male and female mice; the increases were statistically significant at 1800 ppm in both males and females. A significant increase in multiple hepatocellular adenomas were also observed in females at 900 and 1800 ppm and in males at 1800 ppm. Hepatocellular carcinoma was elevated in females at 1800 ppm. While the increase was not statistically significant, it exceeded the historical control range. Combined adenomas and carcinomas were significantly increased in females at 900 and 1800 ppm an in males at 1800 ppm. Stout et al. (2008) note that the histologic appearance of the hepatocellular lesions was consistent with those that develop spontaneously in control mice.

Previous investigations of rodents exposed to MIBK suggest a phenobarbital (PB)‐like signature of changes in the liver‐increased enzyme activity and hepatocellular proliferation that is transient and not sustained, starting after 3‐4 days and lasting as long as 14‐28 days of treatment but no longer (Kolaja et al., 1996; Whysner et al., 1996).

Regarding the mouse liver tumors, the IARC Working Group noted that “there was no evidence that the tumors arose from a cytotoxic‐regenerative cell proliferation mechanism as no overt toxicity occurred in the livers of exposed mice.” The Working Group further explained that “only weak evidence exists that the tumors arose through a receptor‐mediated mechanism, resulting from the induction of enzymes (CYP1A1 and CYP2B) that have been considered to be typical targets of the aryl hydrocarbon receptor and the constitutive activated receptor, respectively (Nebert et al., 2000; Zelko & Negishi, 2000).“ As a consequence, they concluded that the strength of evidence that male and female liver tumors arose through a nuclear receptor mechanism is weak.

In a non-guideline and non-GLP study, the effects of methyl isobutyl ketone (MIBK) on the mouse liver were evaluated (Geter, 2009). Male B6C3F1 mice were implanted with 5-bromo-2’deoxyuridine (BrdU) pumps and then exposed to 0 or 1800 ppm (n=6/group) of MIBK via whole-body inhalation for 6 hours/day for 7 days. In-life assessments included clinical signs and body weights. Mice were euthanized and assessed for clinical chemistry, gene expression analysis of the upper third of the left liver lobe, liver histopathological examination and BrdU proliferation analysis, and liver enzyme activity. There were no treatment-related effects noted for clinical signs, body weights, liver weights, or clinical chemistry assessments. Treatment-related findings included very slight hepatocytes hypertrophy with increased cytoplasic eosinophilia in the centrilobular/midzonal regions of the hepatic lobule which were consistent with increased smooth endoplasmic reticulum and induction of cytochrome P450 enzymes. CYP2B10 transcript levels increased 4-fold and CYP4A10 decreased 5.56-fold. This was verified by increased CYP2B10 enzyme activity (PROD) and hepatocyte proliferation. These responses are commonly observed following activation of constitutive androstane receptor (CAR) and indicate that MIBK may be an agonist ligand for CAR in mice and share a similar mode of action to that of Phenobarbital in mice. The study authors noted that this mechanism of action is not relevant to humans.

To further investigate the MoA for MIBK-induced murine liver tumors, male and female B6C3F1, C57BL/6, and Car/Pxr Knockout (KO) mice were exposed to either 0 or 1800 ppm MIBK for 6 h/d, 5 d/w for a total of 10 days (Hughes et al., 2015). On day 1, mice were implanted with osmotic mini-pumps containing 5-Bromo-2-deoxyuridine (BrdU) 1 h following exposure and humanely euthanized 1 to 3 h following the final exposure. B6C3F1 and FC57BL/6 mice had statistically significant increases in liver weights compared to controls that corresponded with hepatocellular hypertrophy and increased mitotic figures. Hepatocellular proliferation data indicated induction of S-phase DNA synthesis in B6C3F1 and C57BL/6 mice exposed to 1800 ppm MIBK compared to control, no increase was observed in MIBK exposed Car/Pxr KO mice. Liver gene expression changes indicated a maximally-induced Cyp2b10 (Car-associated) transcript and a slight increase in Cyp3a11(Pxr-associated) transcript in B6C3F1 and C57BL/6 mice exposed to 1800 ppm MIBK compared to controls, but not in Cyp1a1 (AhR associated) or Cyp4a10 (PPAR-α-associated) transcripts. Car/Pxr KO mice showed no evidence of activation of AhR, Car, Pxr or PPAR-α nuclear receptors via their associated transcripts.

MIBK induced hepatic effects are consistent with a phenobarbital-like MOA where the initiating events are activation of the Car and Pxr nuclear receptors leading to rodent liver tumors.

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